Pistachio (Pistacia spp.) is a tree nut crop with relatively high salinity tolerance. Currently, limited information exists on its rootstock’s cellular responses to salinity stress, especially in its roots. In this study, we investigated salinity tolerance at cellular level, in two pistachio rootstocks, Pistacia integerrima (PGI) and a hybrid, P. atlantica x P. integerrima (UCB1). Root tip sections were categorized across a developmental gradient according to their xylem development, and their sodium content and suberin deposition were analyzed with fluorescence microscopy. Our data demonstrated a correlation between vacuolar sequestration of sodium ions (Na+) and salinity tolerance in the UCB1 genotype. In addition, UCB1 displayed higher basal levels of suberization in both the exodermis and endodermis that increased further after salinity stress. Notably, the root region immediately distal to the region of secondary xylem initiation showed the highest amount of vacuolar Na+ sequestration, indicating a developmental regulation of this process. Our cumulative data demonstrate that salinity tolerance in pistachio rootstock species is associated with both vacuolar Na+ sequestration and suberin deposition at apoplastic barriers, and both are correlated with a root developmental gradient. These cellular characteristics are phenotypes that can be screened during the selection for salinity tolerant woody plant species.